1. Field of the Invention
The present invention relates to a method for the determination of glucose concentration in blood, especially in serum, such concentration is called the "blood sugar concentration". In particular, the present invention relates to a method for the quick determination of glucose concentration in whole blood with a minimum error.
2. Description of the Related Art
Determination of glucose concentration in blood is required in medical research and treatment of some medical conditions. Various methods for the determination of the glucose concentration in the blood have been proposed and are carried out.
Among these methods, the glucose concentration in the blood is generally determined with a biosensor. Such a method is called the Glucose Sensor Method. In the glucose oxidase biosensor method, a glucose oxidase (GOD) fixed membrane is used in combination with a hydrogen peroxide electrode.
The Glucose Sensor Method is widely used especially in diagnosis and monitoring of diabetes since the glucose concentration is detected with high sensitivity without any pretreatment of blood because of substrate specificity obtained by using the glucose oxidase as part of the detection method.
Such biosensor method used for the determination of the blood sugar concentration in the blood comprises steps of:
separating a supernatant (plasma or serum) from the blood by centrifugation,
diluting the supernatant with a suitable buffer solution,
reacting glucose, oxygen and water in the supernatant with the fixed glucose oxidase,
measuring an amount of hydrogen peroxide produced by the enzyme reaction in terms of an output (current) of the hydrogen peroxide electrode, and
determining a decomposition rate of glucose, that is, a production rate of hydrogen peroxide.
FIG. 1 schematically shows an apparatus used in the biosensor method for the determination of the glucose concentration in the serum. A cell 1 for the determination of the glucose concentration comprises a GOD fixed hydrogen peroxide electrode 2, and a liquid in the cell is thoroughly stirred with a stirrer 3 and a stirring member 4. A buffer solution is supplied in the cell through a valve 6 with a pump 5. After the determination, the liquid in the cell is discharged through a valve 8 with a pump 7. A sample to be determined is supplied in the cell with a sample 9.
The decomposition rate of glucose by the glucose oxidase is proportional to the glucose concentration in the buffer solution. However, the amount of the glucose decomposed is so small that the glucose concentration in the buffer solution is regarded to be constant. Thus, the hydrogen peroxide production rate is constant in a steady state. When the sample is supplied in the buffer solution, the output current from the hydrogen peroxide electrode is, for example, as shown in FIG. 2. The output current of the hydrogen peroxide electrode becomes constant after about 10 seconds from the sample supply.
A calibration curve is beforehand obtained which shows a relation between the glucose concentration and the output current of the hydrogen peroxide electrode after 10 to 20 seconds from the sample supply. Then, the glucose concentration the sample to be measured is obtained as follows: the output current of the hydrogen peroxide electrode with respect to the sample diluted with the buffer solution is measured; and the glucose concentration in the buffer solution which corresponds to the measured output current is read from the calibration curve. The glucose concentration in the diluted sample is converted to the glucose concentration in the undiluted sample by multiplying by the dilution ratio. This determination method is herein called the "Equilibrium Method".
When the glucose concentration in the sample is measured, the sample is usually diluted with the buffer solution as described above. The term "measured glucose concentration" is, hereinafter, intended to mean a glucose concentration which is the glucose concentration in the diluted sample. "Measured glucose concentration" is obtained from the measured output current by reading from a calibration curve plotted from output current measurements of some aqueous glucose solutions of known glucose concentration.
When the curve as shown in FIG. 2, namely, the curve which shows a relation between the output current (I) of the hydrogen peroxide electrode and time (t) is differentiated with respect to time (dI/dt), a curve as shown in FIG. 3 is obtained. A relative maximum value (i.e. a maximum changing rate of the hydrogen peroxide electrode output current) on the curve in FIG. 3 is proportional to the glucose concentration in the buffer solution. Thus, when a relation between the glucose concentration and the relative maximum value of dI/dt has been obtained beforehand as a calibration curve, the measured glucose concentration in a certain sample to be measured is obtained by measuring the relative maximum value of the changing rate of the output current of the hydrogen peroxide electrode immersed in the sample. This method to obtain the glucose concentration in the sample as described above is herein called the "First Differential Method". According to this method, the glucose concentration is obtained after 2 to 3 seconds from the supply of the sample into the buffer solution.
FIG. 4 shows a curve which results from a second order differentiation with time of the curve shown in FIG. 2 (d.sup.2 I/dt.sup.2). A relative maximum value on the curve shown in FIG. is also proportional to the glucose concentration in the buffer solution. Thus, the glucose concentration in the buffer solution can be determined from the relative maximum value as in the First Differential Method. This method as just described above is called the "Second Differential Method". According to this method, the glucose concentration can be advantageously obtained in a shorter time than in the First Differential Method.
Broken lines in FIGS. 2 and 3 and dashed lines in FIGS. 3 and 4 each indicate correspondency of time as shown with arrows.
The glucose concentration obtained by any of the methods as described above is that in a homogeneous solution, for example the buffer solution in which the serum is diluted. Therefore, the serum must be obtained by previously separating blood cells from the blood by centrifugation. It takes about 10 to 15 minutes to centrifugally separate the serum. As long as such separation is required, a quick determination of the glucose concentration is impossible.
The biosensor method is preferably applied to whole blood since the glucose concentration is quickly obtained. The following problems arise in the use of any of the methods described above.
The blood consists of the serum and the blood cells, and the blood cells contain a liquid component therein. The glucose concentration in the blood cells is the same as that outside the blood cells. A solid component of the blood is contributed by the blood cells. The amount of the solid component is generally 25 to 40% of the blood by volume.
For example, in the case where a whole blood sample is introduced into an isotonic buffer solution and then the glucose concentration is measured, glucose in the blood cells transfers into the buffer solution within about 10 seconds to form an equilibrium state in which the glucose concentration in the buffer solution is the same as that inside the blood cells. In this case, not only the serum of the blood but also the liquid component in the blood cells are diluted with the isotonic buffer solution. Then, all of the glucose in the whole blood is measured. However, a true glucose concentration (in which the solid component in the blood is taken into account) cannot be obtained since the ratio of the blood cell volume to whole blood volume is unknown, thus, the true dilution ratio is unknown. Correction of the concentration was proposed in 1980 by WHO (World Health Organization) by the use of an average ratio of the volume of the blood cells to the whole blood volume (blood cell ratio). However, the ratio of the blood cell volume to whole blood volume is highly variable among individuals. Thus, correction based on an average value introduces a rather large error.
When the First Differential Method is employed, equilibrium is reached within a short time, for example after 2 or 3 seconds from the time the sample is supplied. The glucose concentration measured by this method corresponds to that of the buffer solution which contains not only the glucose in the serum but also a small amount of glucose from the blood cells. Also in this case, the true glucose concentration cannot be obtained since the ratio of the volume of the blood cells to the whole blood volume is unknown. A corrected glucose concentration may be obtained using the average blood cell ratio (hematocrit value). Since the hematocrit value varies among individuals, the corrected glucose concentration includes a rather large error.
In the case where the Second Differential Method is employed, not only the glucose in the serum but also a smaller amount of glucose from the blood cells is measured. It is easily understood that the same problems as arise in the First Differential Method arise in this case.
The amount of glucose which is liberated from the blood cells in the Second Differential Method is less than that in the First Differential Method. Although in the method of the present invention, described below, the First Differential Method or the Second Differential Methods is used, the present method is not affected by the amount of liberated glucose since it is very small and the error is canceled when the measured glucose concentration is converted as described below.
As described above, the Glucose Sensor Method is an effective method to quickly measure the glucose concentration itself. However, the centrifugal separation to obtain the serum cannot be omitted as long as the blood cell volume to whole blood volume ratio is unknown. Therefore, the glucose concentration cannot be measured in the whole blood.